Vaccines have been successfully employed for many years in prophylactic compositions for the prevention of infectious disease and more recently in therapeutic compositions for the treatment of cancers and non-infectious diseases.
Traditionally vaccines have been derived from attenuated or killed viral or bacterial pathogens and have proven to be very effective against diseases such as polio virus and Bordetella pertussis. In spite of these successes, there are growing concerns over the safety of such vaccines. This has led to the development of subunit vaccines derived from components of these pathogens or fully synthetic peptide immunogens.
Examples of subunit vaccines include Tetanus toxoid and hepatitis B surface antigen. These antigens are often poorly immunogenic and require adjuvants to improve the immune responses obtained. Well-characterized biologically active compounds such as synthetic peptides are preferred substrates for inducing biological responses, for safety and regulatory purposes. However, these immunogens are not optimal, and induce partial or negligible protective responses in animal models. The synthetic peptides require both stabilization and adjuvantation for the induction of an effective immune response in vivo.
Various methods have been employed to protect synthetic peptide immunogens against degradation in vitro and in vivo, mediated by various processes including chemical and physical pathways.1 (The superscript numbers refers to publications which more fully describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference. The citation of each reference is found at the end of this section.)
Various methods have been employed to improve peptide solubility or protect a peptide against degradation in vivo.2 These generally include simple procedures like modifying the salt concentration and/or the pH of the solution. Peptides have also been chemically modified by conjugation with water soluble compounds like polyethylene glycol (PEG) or polyethylene oxide (PEO) both to improve their aqueous solubility and circulation time in vivo.3 It has been documented that adjuvants derived from PEG or PEO can down regulate the immune system.4 Thus, PEG or PEO modified peptides would not be expected to function effectively as adjuvants. The addition of multiple lysines to add charge to a peptide can improve its aqueous solubility but does not generally result in improved immunogenicity.
The objective of these various strategies is to improve circulation time in vivo or minimize or eliminate immunogenicity problems associated with the physical conditions (e.g. salt, pH, temperature, buffer type) and/or chemical incompatibilities when peptides are employed in a vaccine formulation.
Polyether block copolymers, comprising polycationic polymers, were disclosed by Kabanov et al., U.S. Pat. No. 5,656,6115 for stabilizing polynucleotides or oligonucleotides. The polyether block copolymer-polynucleotide complexes are employed to facilitate the transport of the polynucleotide across a cell membrane for improved biological activity. However, these polynucleotide-polyether block copolymers are not immunogenic and are not suitable as vaccines.
Allcock et al. U.S. Pat. No. 5,562,9096 describes an immunoadjuvant derived from phosphazene polyelectrolytes. The immunoadjuvant was admixed directly with an antigen in solution and may be prepared as microparticles by spray drying a solution of the polymer and the antigen or by a process described by Cohen in U.S. Pat. No. 5,149,543.7 Although, increased adjuvanticity was shown for these systems, there are difficulties in preparing the microparticular compositions due to the cumbersome mechanical processes employed, which would be difficult to scale up for commercial production. Furthermore, the stability of the polymer-antigen complex so formed is highly dependent on salt concentration and pH conditions.
A different approach is described in Moss et al. WO91/040528, wherein a solid vaccine composition is prepared from an antigen, which may be a peptide, a saponin and a polycationic adjuvant such as DEAE-dextran. Vaccines formulated from this combination provided improved longevity, making such combinations suitable for use as implants. However, the antigen must first be chemically conjugated to a carrier molecule and exhaustively purified. The purified antigen-carrier was then combined with a saponin and a polycationic adjuvant to provide a solid composition. This process provides no control over the physical properties, such as particle size, of the product.
Numerous adjuvants and/or depot-based parenteral, mucosal or transdermal delivery systems destined for use with human or veterinary vaccines have been developed to enhance the immune response. These include the use of mineral salts, water-in-oil (w/o)-emulsions, liposomes, polymeric microparticles, nanoparticles and gels/hydrogels.9 A large number of clinical trials employing various (w/o)-emulsion compositions have been conducted.
In spite of this vast body of clinical research, typical parenteral formulations, administered subcutaneously or intramuscularly, are prepared with adjuvants derived from aluminum salts, such as aluminum phosphate or aluminum hydroxide. Alum salts are suitable and effective for many vaccines based on attenuated pathogens, killed pathogens and subunit antigens derived from biological agents. However, the aluminum-based adjuvants are often totally ineffective for synthetic peptide-based immunogens because of the large dose of peptide required and the need of much stronger adjuvantation. The combination of a large dose of immunogen with a weakly adjuvanting alum in a vaccine composition is not ideal as it can lead to immunogen tolerance and reactogenicity, i.e. undesired side reactions, such as swelling and redness at the site of injection.
Freund's complete adjuvant (FCA), a suspension of heat-killed M. tuberculosis mycobacteria in mineral oil containing a surfactant, has been recognized as one of the most powerful adjuvant. However, severe adverse reactions, ranging from minor irritation to lesions and sterile abscesses at the site of injection have been documented. Due to these adverse reactions, FCA has been banned from human and veterinary applications.
Thus, there is a clear need to develop adjuvants which are safe without the toxicological and/or reactogenic problems associated with alum or FCA and can effectively enhance immunogenicity and prolong the effectiveness of peptide immunogens to avoid the problem of tolerance associated with alum. It is also most desirable to develop compositions and methods which can both stabilize a peptide immunogen and adjuvant the immune responses in a single composition.
Jones et al.10 have disclosed two specific CpG oligonucleotides that may be co-administered with a peptide-based malaria vaccine in Aotus monkeys to enhance immune responses. In the Jones study, the ionization point (IP) of the peptide used is 5.96 and has a theoretical zero charge.11 By virtue of its amino acid composition, the peptide used would be effectively uncharged at physiological pH in the aqueous solvent selected. Thus, no complexation can take place with the two CpG oligomers. The resultant mixture when formulated in a w/o-emulsion is expected to be transiently adjuvanted. To achieve a useful level of immunogenicity, multiple injections and a large quantity of adjuvant would be required. Further, the long-term stability of such a composition is questionable. In fact, Jones et al. disclosed that it was necessary to employ a large dose of CpG oligonucleotide, 500 μg per injection. Furthermore, the methods, employed to prepare the w/o-emulsions, cannot be easily scaled up for commercial applications. It is to be noted that Jones et al. taught that different CpG oligomers are useful for different mammalian species. For example, a CpG oligomer, CpG ODN 1826 is mitogenic for mice and a lower primate, but not for chimpanzees or humans and the effect is not predictable.
Krieg et al., U.S. Pat. No. 6,194,388 B112 describes unmethylated CpG oligonucleotides particularly useful for therapeutic applications based on their ability to stimulate immune responses when mixed with an antigen. Krieg et al., U.S. Pat. No. 6,207,646 B113 further describes the use of unmethylated CpG oligonucleotides to redirect a Th2 response to a Th1 response. In both, the effectiveness of the CpG oligomers were shown by B-cell stimulation wherein B-cells were cultured with phosphorothioate modified CpG oligomers. There is no disclosure or suggestion on how the CpG oligomers can be used to provide a stabilized immunostimulatory complex or a vaccine.
Another area of intense interest and research has been focused on methods to formulate synthetic immunogens for alternate delivery routes, such as mucosally, transdermally, or orally. Mucosal immunity is mediated by the induction of secretory immunoglobulin (sIgA) found in external secretions (e.g. intestinal, bronchial or nasal washings). It is believed that transdermal or mucosal delivery of vaccines would be effective against a majority of pathogenic organisms which gain entry via mucosal surfaces. For example, an orally administered cholera vaccine has been shown to be far superior to the parenterally administered analog.14 
Friede et al., WO99/5254915 teaches that vaccine compositions intended for mucosal use can be derived from a combination of an antigen with a polyoxyethylene ether or polyoxyethylene ester as the primary adjuvant. It was suggested that the target antigen may be a synthetic peptide. Friede et al. also suggest the addition of CpG oligonucleotides into the vaccine composition to provide improved responses. They showed that a combination of a polyoxyethylene ether or polyoxyethylene ester with a CpG oligonucleotide can improve mucosal responses when co-administered with an antigen. However, the results showed lack of any adjuvanticity from the various mixtures of CpG ha oligonucleotides with antigen described.
Transdermally administered vaccines represent an area of recent interest. Ideally, devices, i.e., patches or needle-free jet injectors, can be employed to target the intradermal Langerhan cells, i.e., dendritic cells. These specialized cells are responsible for the effective processing and presentation of an immunogen and can be used to directly induce systemic humoral and cellular responses. In some cases, intramuscular immunization was achieved by transdermal methods.16 For example, a recent paper described a diptheria vaccine administered as a patch. Systemic antibodies to diptheria toxoid were found for a variety of compositions when co-administered with adjuvants.17 
Although the prior art has illustrated the potential of various vaccine formulations, there are a number of practical limitations for the development of synthetic peptide-based vaccine formulations for mucosal or transdermal delivery. These include:                1) immunogen degradation by mucosal fluids or secretions and/or proteolytic enzymes at the mucosal surface or within the intradermis;        2) negligible adsorption across the mucosal epithelium or through the intradermal layers; and        3) dilution of the immunogen to a concentration below that required to induce a suitable level of immune responses.        
Few strategies exist which both stabilize and adjuvant a synthetic peptide-based immunogen in a single vaccine composition. Such a composition would be essential for the development of highly efficacious parenteral, mucosal or transdermal vaccines.
It is also desirable to prolong the duration of immunogenic responses in order to reduce the number of administrations required. This would result in improved compliance and reduce the overall cost for vaccination.
Various methods may be employed to adjuvant synthetic peptide-based immunogens, but normally a carrier or depot system is required for effective long-term immunogenic responses. Notable examples include adsorbing the immunogen onto a mineral salt or gel. For example, encapsulating a peptide immunogen within a polymeric matrix (monolithic matrix) or gel, or layering a polymeric material around a peptide immunogen (core-shell) may be an effective strategy. Or, an immunogen may be incorporated in a liposome or vesicular type of formulation, with the immunogen either embedded in the lipid matrix or physically entrapped in the internal aqueous phase. Another strategy may employ a mineral-based, vegetable-based or animal-based oil, with an aqueous solution of the immunogen in various proportions, to prepare a water-in-oil (w/o)-emulsion or a water-in-oil-in-water (w/o/w)-double emulsion18.
Diverse particle sizes, morphologies, surface hydrophobicity and residual surface charge are possible formulation dependent variables for consideration. Control of these parameters is known to be important for the phagocytosis of micron-sized particulates via parenteral administration19, 20 and for the uptake of particulates at specialized M-cells of the Peyers Patches within the intestinal tract21, 22 for oral delivery. Similarly, these parameters have been shown to be important for access to the nasal-associated lymphoid tissue of the nasalpharyngeal tract, a target of intranasal delivery.23, 24 
Krone et al., U.S. Pat. No. 5,700,45925 describes the use of polyelectrolyte complexes in microparticulate form derived from polyacids and polybases, in which the complexing agent is a polymer. Various uses for these complexes, are described and include vaccine compositions comprising antigens or antigenic peptides. Some of the compositions are controlled release formulations employing potentially biodegradable materials. In one of the examples, a method of incorporating an antigen in polyelectrolyte complex microparticles is described. However, the mechanical process described for preparing microparticles by grinding the mixture of 100 μM size particles to about 1-4 μM, is cumbersome. This would not be easily scaled up for commercial production.
Eldridge et al.26 developed polymeric biodegradable microspheres manufactured from poly-D,L-lactide-co-glycolide copolymers for the controlled release of an antigen in vivo. The polymers disclosed to be useful for encapsulating an antigen into microparticles include poly-D,L-lactide, polyglycolide, polycaprolactone, polyanhydrides, polyorthoesters and poly(α-hydroxybutyric acid).
Although the controlled release of an antigen was achieved in the prior art, difficulties were encountered when microparticles were manufactured by methods described. The methods described are difficult to scale-up. Moreover, the exposure of biological materials to organic solvents and mechanical processing can lead to denaturation and low to modest encapsulation efficiencies. Furthermore, hydrophilic antigens are inefficiently encapsulated in the processes described.
Henry, et al., U.S. Pat. Nos. 5,126,141 and 5,135,75127, 28 described aqueous, thermally reversible gel compositions formed from a polyoxyalkylene polymer and an ionic polysaccharide for application to injured areas of the body to prevent adhesion. Rosenberg, et al., WO93/0128629 described the use of the same type of polyoxyalkylene polymers for the local delivery of antisense oligonucleotides to surgically exposed surface of blood vessels for treatment of restenosis. Neither Henry et al. or Rosenberg et al. taught or suggest the use of a gel composition as a vaccine.
Dunn et al., U.S. Pat. Nos. 4,938,763 and 5,702,71630, 31 describe polymeric compositions useful for the controlled release of biologically active materials. A biocompatible solvent was used to prepare solutions or suspensions of antigen for direct parenteral injection, whereupon in-situ gelling results in implant formation. Utility for a variety of antigens including small synthetic peptide-based immunogens was claimed. However, Dunn et al., U.S. Pat. No. 5,702,71631, stated that the controlled release compositions require up to 15% by weight of a gel rate-retarding agent. The retarding agents were added to modulate the gelling rate and were needed for higher entrapment efficiencies for antigens which are easily extracted in vivo. As the solvent extraction is governed largely by diffusion, this presents more of a problem for small synthetic immunogens than for larger sub-unit or protein-based antigens.
Neither U.S. Pat. No. 4,938,76330 nor U.S. Pat. No. 5,702,71631 taught nor suggested synthetic peptide-based immunogen stabilized as an immunostimulatory complex suspended within a biocompatible solvent. Furthermore, neither U.S. Pat. No. 4,938,76330 nor U.S. Pat. No. 5,702,71631 taught nor suggested compositions which are self adjuvanting and can upregulate immune responses in both the priming and boosting phases.
It is an object of this invention to develop stable immunostimulatory complexes from synthetic peptide immunogens and stabilizing molecules, which possess self adjuvanting properties in vivo. It is a further object of the present invention to provide a simple method to stabilize a synthetic peptide immunogen in vitro and in vivo.
It is a still further object of the present invention to provide sustained or controlled release delivery vehicles compatible with these stabilized synthetic peptide-based immunostimulatory complexes.
It is a still further object of the invention to develop formulations using a combination of stabilized synthetic peptide-based immunostimulatory complexes and uncomplexed immunogens in a controlled release delivery system to achieve a synergistic enhancement of the immune responses.